DE3005409A1 - METHOD FOR PRODUCING L-VALINE OR L-LYSINE - Google Patents

Description

HOFFMANN * EITLE & PARTNER

PAT E N TAN WALT E

DR. IMG. E. HOFFMANN (1930-197). DIPL-ING. W.EITLE DR. RER. NAT. K. HOFFMANN. DIPL.-1NG. W. LEHN

DIPL.-ING. K. FOCHSLE DR. RER. NAT. B. HANSEN ARABELLASTRASSE 4 {STAR HOUSE). D-8000 MONCH EN 81 TELEPHONE (089) 911087. TELEX 05-29419 (PATH E)

33087 o / fi

MITSUBISHI PETROCHEMICAL CO., Ltd. Tokyo / Japan

Process for the production of L-valine or L-lysine

The invention relates to a method for fermentative Production of L-valine or L-lysine. L-valine and L-lysine are useful essential amino acids that can be used in medicinal products, dietary foods and the like, and for those having a need for one economical production.

In the production of amino acids by fermentation, saccharides (carbohydrates) are the main starting materials used, but these methods have a number of disadvantages, such as high cost and poor reproducibility on because the raw materials

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are agricultural products. Other disadvantages are the numerous by-products that are formed in the process be, and on the large proportion of in the raw materials contained contaminants, and also the generation of discolored wastewater in the fermentation process- Therefore there is a need for a fundamental improvement in the known procedure.

As an alternative one has the use of hydrocarbons investigated as starting materials, but even these processes have disadvantages because some hydrocarbons are gaseous and others are not soluble in water and therefore their use on an industrial scale is only limited possible.

The object of the invention is to provide an economical process to show the production of L-valine and L-lysine. Associated with this task is to find a method for To show the production of L-valine and L-lysine using easily available and cheap raw materials can.

The invention relates to a process for the production of L-valine and L-lysine, in which ethanol is fermented. According to the invention, a bacterium of the genus Acinetobacter is shown which is able to convert amino acids by fermentation of ethanol to produce.

The inventors proposed the use of ethanol from the raw material for the production of amino acids thoroughly studied. Ethanol is always available and sufficient and inexpensive available and does not have the disadvantages mentioned above.

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It has been found according to the invention that certain bacteria with the ability to produce and accumulate substantial amounts of L-valine and / or lysine, ethanol as a culture medium and as a main source of carbon, and that these bacteria are genus Acinetobacter belong. The invention thus relates to a method for the production of L-valine or L-lysine, the is characterized in that aerobic bacteria of the genus Acinetobacter, which use ethanol, and which in are able to form and accumulate L-valine or L-lysine in a culture medium in which ethanol is the main carbon source is included, cultivated.

The bacteria used according to the invention can all naturally occurring strains, as well as all known strains and all strains that have been artificially mutated and which belong to the genus Acinebacter, provided that they consume ethanol and have the ability to form and accumulate L-valine or L-lysine.

An example of a bacterium that accumulates L-valine or L-lysine from ethanol is Acinetobacter Calcoaceticum YK-1011 available from the Fermentation Research Institute, the Agency of Industrial Science and Technology, Japan, under FERM-P No. 4818 on February 9, 1979.

This bacterium is derived from Acinetobacter Calcoaceticum ATCC 19606 and isolated as the 5-methyl-DL-tryptophan resistant strain and has the same properties as Acinetobacter Calcoaceticum ATCC 19606, with the exception of that it is resistant to 5-methyl-DL-tryptophan is.

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The bacteriological properties of the strain YK-1O11 v / earth is given below and marked with positive (+) or negative (-):

Gram staining Fast acid staining stick shape (pleomorphism) Sporulation motility

Strictly aerobic growth at 22 ⁰ C growth at 42 ° C Indole

Hydrogen sulfide Voges-Proskauer test, methyl red test Viscosity assimilation of:

glucose

Sucrose

Furaarat Acetate Citrate Gluconate Malonate succinate glutamate aspartate L-alanine ß-alanine L-arginine L-leucine decane tridecane

Oxidation of:

L-arabinose +

D-galactose +

Glucose +

Lactose +

D-mannose +

Rhamnose +

D-ribose +

D-xylose + D-fructose
Maltose

Raffinose D-sorbitol
strength
Sucrose
Nitrate reduction:

in succinate nitrate + in casitone nitrate
Liquorice milk:

Acid coagulation +

Reduction -

Catalase, +

Urease + phenylalanine deaminase -

Lysine decarboxylase -

Gelatinase -

Cytochrome oxidase -

Flavin pigmentation +

Resistant to penicillin,

(100 IU) +

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■ - 7 -

Hexadecane - growth in 5%

NaCl broth Kerosene - G + C moles (%) 42

Methanol

Ethanol +

The following is the production of 5-methyl-DL-tryptophan resistant strain of Acinetobacter Calcoaceticum ATCC 19606 explained.

The growth of Acinetobacter Calcoaceticum ATCC 19606 is almost completely inhibited on a plate culture medium prepared by adding 200 µg / ml of 5-methyl-DL-tryptophan to an artificial culture medium containing 2.0 g urea, 7.0 g ammonium sulfate, 0.5 g KH ₃ PO ₄ , 0.5 g K ₃ HPO ₄ , 0.5 g MgSO ₄ '7H ₂ O, 2 mg FeSO ₄ * 7H ₂ O, 2 mg MnSO ₄ · 4-6H ₃ O, 2 mg NaCl, 2 mg ZnSO ₄ * ⁷ H ₃ O, 1 liter of water and 2% v / v ethanol. Derivation from the mutant was carried out by subjecting it to a nitrosoguanidine treatment (nitrosoguanidine 200 µg / ml, 30 ° C, 15 minutes, pH 7.0 with trismaleic acid buffer; EA Adelberg et al, Biochem. Biophys. Res. Comm. 18, 788 (1965)) in a known manner and then cultivated it on a plate culture medium containing 200 μg / ml 5-methyl-DL-tryptophan at 30 ° C. for 3 to 5 days and isolated the colony formed. It goes without saying that the derivation of the mutant is not limited to the above nitrosoguanidine treatment, but can also be effected by ultrared irradiation or treatment with various chemicals.

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Preferred embodiments for carrying out the invention are described below.

Ethanol is used in the culture medium as a carbon source is used, the initial concentration preferably being in the range of about 1 to 5% v / v depending on the one used Trunk lies. With increasing consumption, ethanol is replaced from time to time, with an optimal one Concentration (0.01 to 5% v / v and preferably about 0.01 to 2% v / v), which the growth of the strain or does not inhibit the production of L-valine or L-lysine, adjusts.

The nitrogen source (about 0.01 to 15% v / v) can be below Ammonium sulfate, ammonium nitrate, ammonium phosphate, urea and the like depending on the ability of the strain to use the nitrogen source is selected. Depending on the quantities required by the bacteria (about 10% v / v or less), the Culture medium organic nutrient sources such as amino acids (e.g. glutamic acid, alanine, glycine) corn mash, broth, Yeast extract and the like, inorganic salts (e.g. sulfates or hydrochlorides of Ca, Mg, Na, K, Fe, Ni and Co), Vitamins (e.g. vitamins of the B group, pantothenic acid, benzoic acid and the like) are added.

Typical cultivation conditions are around 20 to 37 ° C, and a pH of about 4 to 10 and preferably a temperature of 25 to 35 ° C and a pH of about 6 to 8. The optimal conditions depend on the particular strain used. Generally, the cultivation takes 2 to 7 days. The cultivation is carried out under aerobic conditions.

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After cultivation, L-valine or L-lysine can be obtained from the liquid in a known manner, for example by means of ion exchange resins, activated carbon, by concentrating and crystallizing and the like. Examples of such methods are shown in JIKKEN NOGEI KAGAKU ( Experimental Agricultural Chemistry ), first volume, pages 284 to 285, Tokyo University, Faculty of Agriculture, Department of Agricultural Chemistry, published by Asakura Shobo (1960).

The invention is described in more detail in the examples.

The quantitative determination of L-valine or L-lysine was carried out by using a microorgano-analytical method performed by Leuconostoc Mesenteroides ATCC 8042 and the values reported are those of which the Amounts of L-valine or L-lysine contained in the culture medium were derived.

EXAMPLE

10 ml of a precultivation medium containing 2.0 g urea, 7.0 g ammonium sulfate, 0.5 g KH ₂ PO ₄ , 0.5 g K ₂ HPO ₄ , 0.5 g MgSO ₄ '7H ₂ O, 0.5 g of yeast extract, 0.5 g of casamino acid, 2 mg of FeSO ₄ -7H ₂ O, 2 mg of NaCl, 2 mg of CaCl ₂ '2H ₃ O and 1 l of tap water were poured into a large test tube with an internal diameter of 24 mm and treated for 10 minutes 120 ⁰ C sterilized. 0.2 ml of ethanol was added thereto under aseptic conditions, and then Acinetobacter Calcoaceticum YK-1011 was inoculated and shake cultivation was carried out at 30 ° C for 2 days. Then 10 ml of the same culture medium as in the preculture medium was placed in a large test tube with an inner diameter of 24 mm

- 10 -

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and sterilized at 120 ° C for 10 minutes and then were 0.2 g calcium carbonate which had been sterilized by dry heating and then 0.2 ml ethanol was added and then 0.2 ml of the preculture liquid inoculated and a shaking culture carried out at 30 ° C. for 7 days. Ethanol was used to the extent that v / how it was consumed, added. Care was taken that the ethanol concentration did not exceed 3% v / v. 7 days after the start of the cultivation, it was found that 600 mg / 1 L-valine had accumulated. 100 ml of the culture liquid was centrifuged to separate the cells and the supernatant liquid was passed through a strongly acidic cation exchange resin (Diaion SK-IB, in the acid form), adsorbing L-valine and then 0.5 η ammonia water was used to elute L-valine in a known manner- The L-valine Fractions containing were concentrated, decolorized using activated charcoal and cold ethanol was added to give crude crystals of L-valine. Used under similar cultivation conditions Acinetobacter Calcoaceticum ATCC 19606 so could not Formation of L-valine can be determined.

EXAMPLE

10 ml of a preculture medium (2.0 g urea, 7.0 g ammonium sulfate, 0.5 g KH ₂ PO ₄ , 0.5 g K ₂ HPO ₄ , 0.5 g MgSO ₄ -7H ₂ O, 0.5 g Yeast extract, 0.5 g casamino acid , 2 mg FeSO ₄ ¹ 7H ,, 0, 2 ItRMnSO, -4-6H ^ O, 2 mg ZnSO, * 7H ,, 0, 2 mq NaCl, 2 iriq CaCl ₂ '2H ₂ O, 200 ug biotin, 100 ug thiamine hydrochloride and 1 l tap water) were poured into a large test tube with an inner diameter of 24 mm and allowed Ί0 minutes

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Sterilized at 120 ° C and then aseptically 0.2 ml Ethanol was added and Acinetobacter Calcoaceticum YK-1011 was inoculated and a shaking culture was carried out at 30 ° C. for 2 days carried out. Then 10 ml of the same culture medium as the preculture medium were placed in a large test tube Cast with an inner diameter of 24 mm and sterilized for 10 minutes at 120 ° C and 0.2 g of dry Heat sterilized calcium carbonate and 0.2 ml of ethanol and then 0.2 ml of the preculture liquid inoculated and subjected to a shaking culture at 30 ° C. for 7 days. Ethanol was used up as it was consumed was supplemented. Care was taken that the concentration of ethanol did not exceed 3% v / v. 7 days after Starting the cultivation, a culture liquid containing L-lysine in an amount of 6 g / l was obtained.

50 ml of similarly cultured collected culture liquids were used to separate the cells centrifuged. The supernatant was absorbed by a strongly acidic ion exchange resin Diaion SK-1B sent by L-lysine. Then, 5% v / v ammonia water was passed through to elute the absorbed L-lysine the ion exchange resin was added and the eluate was concentrated under reduced pressure. Became the concentrate Added hydrochloric acid, upon cooling, crystals of L-lysine hydrochloride dihydrate in an amount of 210 mg.

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Claims (6)

HOFFMANN · EITLJS Ä PARTNEIi PATENTANWÄLTE DR. ING. E. HOFFMANN (1930-1976) DIPL-tNG. V / .EITLE · D R. RER. NAT. K. MOFf MANN · Dl PL.-ING. W. LEHN DIPL.-ING. K. FOCHSLE DR. RER. NAT. B. HANSEN ARABELLASTRASSE 4 (STERN HAUS) D-8000 MO N CH EN 81. TELEPHONE (08S>) 911087. TELEX 05-29619 (PATHE) 33 087 o / fi MITSUBISHI PETROCHEMICAL CO., Ltd. Tokyo / Japan Process for the production of L-valine or L-lysine Patent claims: 1. A process for the preparation of L-valine or L-lysine, characterized in that one aerobic a bacterium from the genus Acinetobacter that consumes ethanol and has the ability to produce L-valine or L-lysine manufacture and accumulate, in a culture medium in which ethanol is the main carbon source, cultured with the formation and accumulation of L-valine and / or L-lysine. 2. The method according to claim 1, characterized in that that the bacterium is A. Calcoaceticum YK-1011 = 3. The method according to claim 1, characterized ζ e i ch η e t that the bacterium A. Calcoacettikum ATCC 19606 is. 030034/0773 4. The method according to claim 1, characterized ge indicates that the method of manufacture of L-valine is applied. 5. The method according to claim 1, characterized ge indicates that the process for the production of L-lysine is used. 6. The method according to claim 3, characterized ge indicates that the bacterium is obtained by treatment with nitrosoguanidine. 3U034 / 077 3